Purification and characterization of the soluble form of processing α-glucosidase I from saccharomyces cerevisiae
| Autor: | Faridmoayer, Amirreza |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2005 |
| Druh dokumentu: | Text |
| Popis: | Saccharomyces cerevisiae processing a-glucosidase I (Cwh41p) is a type II membrane-bound glycoprotein encoded by CWH41. This enzyme regulates the first trimming step in the A-glycosylation pathway and may play an important role in glycoprotein biosynthesis and quality control in the endoplasmic reticulum (ER). Despite its importance, there is a limited understanding of the structure, functional residues, and mechanism of aglucosidase I. Therefore this thesis was focused on establishing a robust purification method for the soluble form of yeast a-glucosidase I, identifying the catalytic domain, and determining structural or functional amino acid residues. The soluble form of a-glucosidase I was purified to 95% homogeneity using a combination of ammonium sulfate precipitation, anion-exchange, lectin affinity, and sizeexclusion chromatographies. The molecular mass of soluble a-glucosidase I was 98 kDa by SDS-PAGE. The purification method was improved by cultivation of transformed yeast in a fermenter and using a deoxynojirimycin (DNM)-based column. This method reproducibly yielded 40 pg of pure enzyme per gram of wet biomass with no detectable contamination by other aryl a- and P-glucosidases. Cleavage between Ala24 and Thr25 of the transmembrane domain of Cwh41p released the soluble activity and this fragment was shown to be glycosylated. A luminal 37 kDa non-glycosylated polypeptide was isolated as the smallest active fragment from endogenous and trypsin hydrolysis of the soluble a-glucosidase I, using DNM-based resins. The hydrolysis sites were determined to be between Arg521 and Thr522 for endogenous proteolysis and Lys524 and Phe525 for the trypsin hydrolysis. This 37 kDa polypeptide was 1.9 times more active than the 98 kDa protein when assayed with the synthetic trisaccharide. Site-specific chemical modification of the soluble a-glucosidase I from yeast using diethylpyrocarbonate, tetranitromethane and 3-(3-(dimethylamino)propyl)carbodiimide revealed that histidine, tyrosine and carboxylic acid residues are involved in a-glucosidase I activity, as these residues could be protected from modification using the competitive inhibitor DNM. DNM could not prevent inactivation of enzyme treated with N - bromosuccinimide used to modify tryptophan residues. Functional expression of truncated forms of Cwh41p was also investigated. Only Cwhtlp (E35- F833) was expressed as a catalytically active soluble fragment. Cwhtlp was isolated as a 94 kDa non-glycosylated polypeptide with a specific activity (3600 U/mg of protein) comparable to the soluble a-glucosidase I (3000 U/mg of protein). These results suggest that the Ml-128 region, containing the predicted N-terminal cytosolic segment and transmembrane domain, of Cwh41p likely carries an ER-targeting signal sequence and is not important for protein folding. Alignment of orthologs indicated that six highly conserved carboxylic acid residues resided within the putative catalytic region of yeast a-glucosidase I. Substitution with A la for E580 and D584 of Cwhtlp (E613 and D617 of Cwh41p), that are situated at the corresponding proposed binding motif of the mammalian enzyme, resulted in undetectable aglucosidase I activity. Furthermore, mutants were expressed at considerably lower concentrations than Cwhtlp. These findings suggest that conserved E613 and D617 may play an important functional or structural role in enzyme activity. In conclusion, results of this thesis indicated that the soluble a-glucosidase I is a proteolytic product of Cwh41p and can be functionally expressed without undergoing Nglycosylation. Moreover, the catalytically active fragment (F525-F833) can be isolated from the soluble a-glucosidase I but it can not be expressed alone suggesting an integral structural function for the non-catalytic region (E53-F525). Also, chemical modification results suggest that the potential binding residues are more conserved between yeast and plant, rather than yeast and mammalian a-glucosidase I. Finally, the conserved E613 and D617 may play an important role in yeast a-glucosidase I activity. Land and Food Systems, Faculty of Graduate |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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